JIPB Journal of  IntegrativePlant Biology Invited Expert Reviewhttps://doi.org/10.1111/jipb.13206
Here comes the sun: How optimization
of photosynthetic light reactions can
boost crop yieldsOO
Julia Walter1 and Johannes Kromdijk1,2*
1. Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
2. Carl R Woese Institute for Genomic Biology, University of Illinois Urbana‐Champaign, Urbana Illinois, 61801, USA
*Correspondence: Johannes Kromdijk (jk417@cam.ac.uk)
Julia Walter Johannes Kromdijk
ABSTRACT
Photosynthesis started to evolve some 3.5 bil-
lion years ago CO2 is the substrate for photo-
synthesis and in the past 200–250 years, at-
mospheric levels have approximately doubled
due to human industrial activities. However,
this time span is not sufficient for adaptation
mechanisms of photosynthesis to be evolutio-
narily manifested. Steep increases in human
population, shortage of arable land and food,
and climate change call for actions, now.
Thanks to substantial research efforts and ad-
vances in the last century, basic knowledge of
photosynthetic and primary metabolic proc-
esses can now be translated into strategies to
optimize photosynthesis to its full potential in
order to improve crop yields and food supply
for the future. Many different approaches have
been proposed in recent years, some of which
have already proven successful in different
crop species. Here, we summarize recent ad-
vances on modifications of the complex net-
work of photosynthetic light reactions. These
are the starting point of all biomass production
and supply the energy equivalents necessary
for downstream processes as well as the
oxygen we breathe.
Keywords: bioengineering, crop improvement, electron transfer,
light reactions, photosynthesis, photosystem, stress tolerance
Walter, J., and Kromdijk, J. (2022). Here comes the sun: How
optimization of photosynthetic light reactions can boost crop
yields. J. Integr. Plant Biol. 64: 564–591.
INTRODUCTION—WHY DO WE
NEED CROPS WITH INCREASED
YIELDS?
Crops and farming have sustained human existence formore than 11,000 years (Murphy, 2007). The growing
world population is currently projected to reach 10.87 billion
people by the end of this century in 2100 (Figure 1A, data
from the Food and Agriculture Organization [FAO, https://
www.fao.org/faostat/]) and requires considerably increased
food production. This is a major challenge as agricultural land
becomes more and more limited. In many northern latitude
countries, agricultural areas have not further expanded in the
past 30 years or have even declined somewhat (Figure 1B,
data from FAO, see also Ramankutty et al., 2018). However,
in many tropical areas, agricultural land use has increased by
up to 20% in the past 30 years, with concomitant decreases
in forest area. Up to 40 hectares of forest are being cleared
every minute to generate more arable land in order to pro-
duce more food and feed for animals. This has led to a more
than 50% loss of rainforests to date (Figure 1C, data from
FAO; FAO and UNEP, 2020).
Trees absorb and fix enormous amounts of solar radiation
and carbon dioxide, making a major contribution to mitigate
against global warming and climate change, and serve as
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
© 2022 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany,
Chinese Academy of Sciences.
reservoirs for the freshwater we drink. To tackle deforestation
and preserve nature, alternative approaches need to be devel-
oped to improve productivity of the farmland that is currently
available. One promising target is the chemical process that
sustains all life on Earth, called oxygenic photosynthesis. Plants
and photosynthetic micro‐organisms fix carbon dioxide from the
atmosphere and convert it into sugars and organic biomass,
using the absorbed light energy from the sun and water from the
soil, and releasing molecular oxygen as a by‐product (Figure 2).
The predominant view up to two decades ago was that
photosynthetic efficiency had reached its maximum capacity
and could not be further improved in crops due to sink
limitations, and the subject still sparks scientific debate
(discussed in Long et al., 2006; Sinclair et al., 2019; Araus et al.,
2021; Paul, 2021). However, studies simulating possible future
increases in atmospheric carbon dioxide (CO2) concentrations
have clearly indicated that photosynthesis can indeed be en-
hanced and result in improved crop yields (Ainsworth and Long,
2021), which has been further established over the years (Zhu
et al., 2010; Parry et al., 2011; Gu et al., 2014; Long et al.,
2015; Ort et al., 2015; Yin et al., 2015, 2021a, 2021b; Kromdijk
and Long, 2016; Simkin et al., 2019; Wu et al., 2019).
In recent years, different strategies to improve crop yields
have been proposed and reviewed, such as: (i) introducing
photorespiratory bypasses (Betti et al., 2016; Hagemann and
Bauwe, 2016; South et al., 2018; Eisenhut et al., 2019; López‐
Calcagno et al., 2019; Maurino, 2019; Shen et al.,
2019; Khurshid et al., 2020; Wang et al., 2020; Abbasi et al.,
2021); (ii) introducing algal/cyanobacterial carbon concen-
trating mechanisms (McGrath and Long, 2014; Rae et al.,
2017; Long et al., 2018; Atkinson et al., 2020; Hennacy and
Jonikas, 2020; Chen et al., 2021; Rottet et al., 2021); (iii) in-
troducing the C4 photosynthesis pathway into C3 plants
(Ermakova et al., 2020, 2021a); (iv) improving mesophyll
conductance (Hanba et al., 2004; Xu et al., 2019; Lundgren
and Fleming, 2020; Ermakova et al., 2021c); (v) modifying
metabolic processes (Rossi et al., 2015; South et al., 2019);
and (vi) modifying circadian rhythms and introducing chro-
nocultures (Steed et al., 2021). During the second half of the
20th century, the Green Revolution led to improved grain
yields through conventional breeding techniques and im-
proved pest/disease control. Nevertheless, photosynthesis
still typically performs at a four‐ to five‐fold lower efficiency
Figure 1. Global developments calling for solutions for improved
crop yields
(A) Annual world population (in billion people) recorded from 1950 to 2020
with future predictions from 2021 to 2100. (B) Agricultural land use
development and (C) forest land development per continent over the past
30 years. The shares of annual agricultural land use and forested land
(in %) per total land area was determined for each area. All data were
obtained from the Food and Agriculture Organization (FAO) in October–
December 2021. Agricultural land use includes both crop and pasture
land. Timeseries data for Asia and Europe were started at 1993 to avoid
the discontinuity in 1992 due to the end of the USSR. Groupings by
continent or sub‐continent follow the FAO country groupings. The data for
group “South America” also includes the Caribbean countries. The data for
group “Asia” combines FAO country groups for central, eastern, southern,
and western Asia.
Figure 2. Schemes of terrestrial and marine photosynthesis
Carbon dioxide (CO2) from the air and water (H2O) from the soil are taken
up by land plants and converted into sugars and biomass using the light
energy of the sun (according to the equation 6 CO2+6 H2O↔C6H12O6+6
O2). As a photosynthetic by‐product, molecular oxygen (O2) is released
into the air. Percentages indicate the proportions of total sequestered CO2
and released O2 by terrestrial and marine photosynthesis (source: Food
and Agriculture Organization https://www.fao.org/3/y0900e/y0900e06.
htm, World Ocean Review).
Optimizing light reactions to improve crop yieldJournal of Integrative Plant Biology
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than its theoretical maximum (Long et al., 2015; Ort et al.,
2015). Photosynthetic light use efficiency is a major deter-
minant of the conversion efficiency of absorbed light energy
into biomass. Only 50% of incident solar radiation (wave-
lengths between 400 and 740 nm) can be actively used to
drive photosynthesis. Further energy losses occur due to
light reflectance from the leaf, light absorption by non-
photosynthetic pigments, dissipation of excess light energy
as heat, thermodynamic limits, carbohydrate biosynthesis,
photorespiration, and respiration. This leaves a theoretical
maximum of about 5% of total irradiance that is converted
into biomass. However, in the field, photosynthetic efficiencies
normally only reach 1%–2% on the individual plant level because
of light saturation of the photosynthetic machinery at about
25%–50% of full sunlight and activity of energy‐dissipating
photoprotective mechanisms at higher light intensities (Long
et al., 2006; Zhu et al., 2008, 2010). At the canopy level this
results in photosynthetic efficiencies of about 2.2% under well‐
managed conditions (Yin and Struik, 2015). Calculating these
theoretical efficiencies highlights the importance of crop mod-
eling to consider further routes for crop improvement (Wu et al.,
2019; Yin et al., 2021a, 2021b). Strategies to overcome these
limitations include optimization of the canopy and leaf archi-
tecture (Tholen et al., 2012; Drewry et al., 2014; Mathan et al.,
2016; Song et al., 2013, 2016; Xiao et al., 2016; Slattery and Ort,
2021) as well as the photosynthetic light‐dependent and light‐
independent reactions. While the photochemical light‐
dependent reactions involve harvesting of excitation energy
from sunlight to produce the energy carriers nicotinamide ad-
enine dinucleotide phosphate (reduced form) (NADPH) and
adenosine triphosphate (ATP), in the light‐independent re-
actions these energy carriers are then used to fix carbon di-
oxide via the enzyme ribulose‐1,5‐bisphosphate carboxylase/
oxygenase (Rubisco) into C3 sugars and regenerate the Ru-
bisco substrate ribulose‐1,5‐bisphosphate (RuBP; Calvin–
Benson–Bassham cycle). Both Rubisco activity and the rates of
regeneration reactions have been targeted for improvement,
which have proven very successful in recent years (Lefebvre
et al., 2005; Kurek et al., 2007; Kumar et al., 2009; Rosenthal
et al., 2011; Parry et al., 2013; Whitney et al., 2015; Driever
et al., 2017; Salesse‐Smith et al., 2018; Simkin et al.,
2015, 2017a, 2019; Scafaro et al., 2019a; López‐Calcagno
et al., 2020; Iñiguez et al., 2021), with more promising research
under way (Scafaro et al., 2019b; Degen et al., 2020).
This review focuses on the photosynthetic light‐
dependent reactions, the optimization of which is relevant
to improving both C3 and C4 photosynthesis in plants and
photosynthetic micro‐organisms (Leister, 2012; Ruban,
2015; von Caemmerer and Furbank, 2016; Cardona et al.,
2018; Simkin et al., 2019; Batista‐Silva et al., 2020;
Ermakova et al., 2021b; Sales et al., 2021; Santos‐Merino
et al., 2021). Modeling studies have suggested that im-
proving the quantum yield and electron transport capacity
have a greater potential for increasing the productivity of
crops than other photosynthetic mechanisms, such as
improving Rubisco activity (Gu et al., 2014; Yin and Struik,
2015, 2021a; Wu et al., 2019). In the past couple of years,
numerous novel approaches to improve photosynthetic
light reactions have been reported. This review presents
a synthesis and highlights the potential of these
approaches to boost crop yields.
THE “SOLAR PANELS” OF THE
PLANT CELL—THE LIGHT
REACTIONS OF PHOTOSYNTHESIS
Solar panels are an increasingly popular choice for gen-
erating “home‐made” sustainable energy and circumvent the
use of fossil fuels (International Energy Agency, 2021). The
process of light energy conversion into electricity, called
photovoltaic effect, has been translated into solar cells with
light conversion efficiencies of around 20%–50% (Geisz
et al., 2020). A similar effect can be observed in nature in
photosynthesizing organisms. Here, the light‐harvesting
complexes together with the photosystems act in series to
absorb energy from sunlight and fuel electron transfer and
subsequent redox reactions in the thylakoid membranes,
resulting in the generation of chemical energy which fuels the
production of biomass. However, the photosynthetic light‐to‐
biomass conversion efficiency is more difficult to estimate
than the light‐to‐electricity conversion efficiency in solar cells.
Nevertheless, efforts have been made to determine the the-
oretical photosynthetic efficiency in plants on the individual
plant level as 4.6% and 6.0% (Zhu et al., 2010) for C3 and C4
plants, respectively. However, actual photosynthetic effi-
ciencies can be as little as 1%–3.5%/4.3% in C3/C4 plants
(Zhu et al., 2010; Blankenship et al., 2011). But how exactly
does the light‐harvesting “solar panel” of the plant cell op-
erate when a photon hits the leaf and initiates photosyn-
thesis? And how has it developed during the course of evo-
lution?
As the name suggests, light‐harvesting complex (LHC)
proteins absorb light energy from the sun. The LHC pro-
teins are located in the thylakoid membranes in close
proximity to the photosystems PSII and PSI and act as a
funnel, channeling the absorbed light energy, also called
excitation energy, toward the photosystems' reaction
center chlorophylls P680 (PSII) and P700 (PSI). There,
electrons become excited by reaching a higher energy
level (termed “charge separation”) and move toward
electron acceptors, thus initiating a series of electron
transfers in the thylakoid membrane between the two
photosystems. Charge separation in PSII leads to oxi-
dized P680+ and reduction of the stable electron ac-
ceptors plastoquinone (PQ) A and B (QA and QB, PQ pool)
via the unstable intermediate pheophytin. P680+ is a very
strong oxidant and extracts an electron from water, which
is split into protons (H+) and molecular oxygen (O2) at the
Oxygen‐Evolving Complex in PSII. Further down the line,
doubly reduced QB accepts two H
+ from the stroma,
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forming the mobile electron carrier plastoquinol (PQH2),
which passes on the electron to the membrane‐
embedded cytochrome b6f complex. From there, the
electron either travels back to the PQ pool via the Q‐cycle
or further toward the electron gap in PSI (P700+) via a
mobile carrier in the thylakoid lumen, which is called
plastocyanin. Within PSI then, excited electrons released
from P700 using harvested light energy, are accepted by
phylloquinone followed by electron transfer via three iron‐
sulfur (Fe‐S) clusters to ferredoxin (Fd) and the protein Fd‐
NADP+‐reductase (FNR) which regenerates NADP+ to the
reducing agent NADPH. At the same time, the ultimate
energy carrier ATP is produced upon acidification of the
thylakoid lumen. During linear electron transfer (LET), a H+
gradient across the thylakoid membrane is established.
This electrochemical force (proton motive force—pmf)
drives ATP synthesis via the membrane‐spanning ATP
synthase complex, which generates ATP from adenosine
diphosphate (ADP) and inorganic phosphate (Pi) on the
stromal side. The NADPH and the ATP produced
by photosynthetic electron transport are essential to drive
the Calvin–Benson–Bassham cycle for atmospheric
CO2 fixation and the production of sugars used for
biomass biosynthesis, and for other metabolic processes
in the chloroplast (for a review see Stirbet et al.,
2019; Figure 3).
The more sunlight, the better? How photoprotective
mechanisms safeguard the light reactions from
excessive sunlight
In nature, sunlight is a very variable resource. On a sunny
day, plants can experience fluctuations in light exposure
and spectral features when clouds cover the sun, for in-
stance, or other plants/leaves move in the wind and shade
the canopy below. Thereby, blue and red wavelengths are
absorbed by the upper canopy, depleting the light of
wavelengths that can be captured by the LHC proteins and
drive photosynthesis in the lower canopy. Dynamic light
conditions occur on a seasonal level, a daily level, and as
cloud‐ and sunflecks, which can fluctuate rapidly and last
seconds to minutes (Morales and Kaiser, 2020).
Harvested light energy does not always lead to electron
transfer but can also be dissipated via other routes. If the
sunlight is too strong (termed “high light”), it can be harmful
to the plant. Photosynthesis has several rate‐limiting steps,
such as the regeneration of the Rubisco substrate RuBP as
Figure 3. Overview of light reactions in the thylakoid membrane
In the thylakoid membrane system inside the chloroplast, two pigment–protein photosystems (PSII and PSI) operate in series in order to generate the
energy equivalents nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) and adenosine triphosphate (ATP). Absorbed excitation energy
is channeled by light‐harvesting complexes LHCII and LHCI toward the reaction centers of both photosystems (P680 in PSII and P700 in PSI), where an
electron is liberated and passed along several electron acceptors down the linear electron transfer (LET) chain (blue arrows). Downstream of PSII, the
plastoquinone/plastoquinol (PQ) pool transfers electrons to the cytochrome b6f complex (cyt b6f) and further to PSI via plastocyanin (PC). In PSI, several
iron‐sulfur clusters (Fx, FA, FB) are the primary electron acceptors which then reduce ferredoxin (Fd). Lastly in LET, the Fd‐NADP+ reductase (FNR) is
released from its possible anchor thylakoid rhodanase‐like (TROL) protein, which contains a rhodanese‐like motif (RHO) in the lumen and a membrane
recruiting motif (MRM) in the stroma, and regenerates NADPH by oxidation of Fd. Simultaneously in PSII, oxidized P680+ is reduced by an electron deriving
from the splitting of water (H2O) at the oxygen‐evolving complex, which also releases molecular oxygen (O2) and protons (H+). Protons are also transported
across the thylakoid membrane by cyt b6f and the NADH dehydrogenase‐like 1 (NDH‐1) complex (orange arrows) in order to fuel ATP production at the ATP
synthase complex. Both NADPH and ATP are then metabolized in the Calvin–Benson–Bassham (CBB) cycle for carbon fixation. In case of overexcitation of
the LET chain, AET routes are activated downstream of PSI (magenta dashed arrows), including cyclic electron transfer via the Proton Gradient Regulation
5 (PGR5)/PGR5‐like photosynthetic phenotype 1 (PGRL1) or the NDH‐1 complexes. NDH‐1 also diverts excess electrons to the Plastid Terminal Oxidase
PTOX, which reduces O2 to H2O. Photoprotection via qE‐type nonphotochemical quenching (NPQ) involves the PSII subunit S protein PsbS, which senses
the acidification of the lumen upon high light exposure via protonatable residues and initiates the rearrangement of the LHCII complexes, thus inducing the
dissipation of excess excitation energy or NPQ. In parallel, the xanthophyll cycle is also activated by the lumen pH, inducing the reversible conversion of
violaxanthin (yellow pigment) into antheraxanthin and zeaxanthin (red pigment) in light via the violaxanthin de‐epoxidase (VDE). In dark, the xanthophyll
cycle is then reversed by zeaxanthin epoxidase (ZEP).
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electron sink in the Calvin–Benson–Bassham cycle as well as
the activity of the cytochrome b6f complex and the ATP
synthase in the light reactions (Farquhar et al., 1980; Price
et al., 1998; Hajirezaei et al., 2002; Sage et al., 2008; Rott
et al., 2011; Yamori et al., 2011; Hasan and Cramer,
2012; Magyar et al., 2018). These bottlenecks can result in a
jam of electrons in the thylakoid membrane when the ca-
pacity of light‐harvesting exceeds the capacity of CO2 fix-
ation. Under these conditions, the formation of chlorophyll
triplet states from singlet excited chlorophylls increases.
Triplet chlorophyll can readily react with molecular oxygen
and form the harmful reactive oxygen species (ROS) singlet
oxygen. This highly reactive state of oxygen can specifically
damage the photosynthetic protein complexes in the thyla-
koid membrane, oxidize plasma membrane lipids and react
with nucleic acids (Krieger‐Liszkay, 2005; Di Mascio et al.,
2019; Khorobrykh et al., 2020). Particularly, the PSII core
protein D1 is prone to oxidative photodamage upon long‐
term high light exposure, causing a transient downregulation
of PSII efficiency, termed photoinhibition of PSII (Aro et al.,
1993; Long et al., 1994; Murata et al., 2007; Guidi et al.,
2019). Since plants are sessile organisms, they are not able
to change location to avoid unfavorable conditions and have
developed numerous photoprotective mechanisms during
the course of evolution. One such mechanism is via
nonphotochemical dissipation of excess absorbed energy as
heat (nonphotochemical quenching [NPQ]).
In addition to photochemistry and NPQ, excited chlorophylls
can also return to the ground state via fluorescence, that is,
emission of a red‐shifted photon. While fluorescence emission is
not an appreciable energy flux, analysis of fluorescence
quenching allows estimation of energy dissipation via photo-
chemistry and NPQ. NPQ measured by fluorescence quenching
analysis is a collective term that includes several different com-
ponents for the avoidance responses of photodamage. These
actually do not dissipate excess energy as heat but aim at de-
creasing light absorption and optimizing electron distribution,
such as chloroplast photorelocation, redistribution of LHCII be-
tween the photosystems (state transitions) and photoinhibitory
break‐down of D1 in PSII (for a comprehensive overview and
depiction see Malnoë, 2018; Messant et al., 2021). Energy dis-
sipation in the LHCs via NPQ consists of different components
with contrasting response times. The fastest component of ac-
tual heat dissipation mechanisms is called energy‐dependent
quenching (qE) (Wraight and Crofts, 1970, reviewed in Ruban,
2016, depicted on the left‐hand side in Figure 3). qE is triggered
by lumen acidification, with the sensitivity of the response highly
dependent on the PSII subunit S protein (PsbS) in higher plants.
This protein is considered the main player in the induction of
photoprotective mechanisms and acts as a pH sensor in the
thylakoid membrane. Upon illumination and thus acidification of
the thylakoid lumen, PsbS undergoes a conformational change
and activates quenching of excess absorbed light energy. A very
likely location of the active quenching site in land plants are the
LHC antenna proteins, which are involved in light‐harvesting as
well as photoprotection of the photosystems' reaction centers.
LhcA1‐4 proteins form heterodimeric antennae around PSI,
whereas LhcB1‐3 assemble either into strongly PSII‐bound
S‐homotrimers of LhcB1 or moderately and loosely PSII‐bound
M‐ and L‐heterotrimers of LhcB1‐3. These major antennae of
LHCII trimers are connected to the PSII core proteins via the
monomeric minor antennae LhcB4‐6. While LhcB4 (CP29) and
LhcB6 (CP24) form heterodimers, connecting the M‐LHCII
trimers to the PSII core via the CP47 protein, LhcB5 (CP26)
connects the S‐LHCII trimers to the PSII core via the CP43
protein, thus channeling the absorbed sunlight energy from the
LHCII trimers to the PSII reaction center via the monomeric an-
tenna proteins (Figure 4A,B). Although the exact NPQ mecha-
nisms are not known yet, it seems clear that PsbS dimers sense
the change in lumenal pH upon light exposure with two proto-
natable lumen‐exposed glutamate residues per monomer (Li
et al., 2004). Subsequently, the rearrangement of the LHCII an-
tennae is initiated, potentially via PsbS monomerization and in-
teractions with the CP29 and LhcB1 proteins. In a putative model
for qE, conformational changes are also induced at the super-
complex level, where M‐LHCII trimers are released from the PSII‐
LHCII supercomplexes for the formation of LHCII aggregates in
the thylakoid membrane, forming the putative quenching site Q1
(Bergantino et al., 2003; Teardo et al., 2007; Betterle et al.,
2009; Miloslavina et al., 2011; Dall'Osto et al., 2017; Kress and
Jahns, 2017). Besides the acidification of the lumen pH, another
prerequisite of qE is the activation of the reversible xanthophyll
cycle and binding of the xanthophyll zeaxanthin to major and
minor LHCII proteins (Niyogi et al., 1997; Bassi and Caffarri,
2000; Ballottari et al., 2012). Under dark and low light conditions,
LHCII proteins predominantly bind violaxanthin, whereas upon
exposure to high light, violaxanthin is released into the thylakoid
membrane, converted into zeaxanthin via the intermediate xan-
thophyll antheraxanthin by the enzyme violaxanthin de‐
epoxidase (VDE), and reinserted to induce NPQ and protect the
cell components from oxidative stress (Havaux et al.,
2007; Johnson et al., 2007; Dall'Osto et al., 2010). Zeaxanthin is
reconverted into violaxanthin by the stromal enzyme zeaxanthin
epoxidase (ZEP) upon light‐to‐dark transitions. Zeaxanthin is also
involved in a PsbS‐independent NPQ mechanism, called qZ
(zeaxanthin‐dependent quenching) for which induction and re-
laxation are correlated with zeaxanthin formation and depletion
on a time scale of several minutes (Dall'Osto et al., 2005; Nilkens
et al., 2010). Whereas further long‐term quenching forms were
previously collectively termed qI (inhibitory quenching), this pa-
rameter is currently subject to further molecular dissection based
on the factors involved. For example, a long‐term form of NPQ (in
the range of hours), called qH, has recently been discovered to
occur in the LHCII trimers, involving the plastid lipocalin LCNP
and its regulators SOQ1 and ROQH1 (Malnoë et al., 2018; Am-
stutz et al., 2020; Bru et al., 2020, 2021; Yu et al., 2021).
In addition to NPQ, several alternative electron transfer (AET)
routes become active in response to stress in order to prevent
the LET chain from overreduction. To maintain an optimal ATP/
NADPH ratio for metabolic processes under such conditions,
electrons are rerouted from Fd back to the PQ pool potentially
via the Proton Gradient Regulation 5 (PGR5)/PGR5‐like
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photosynthetic phenotype 1 (PGRL1) complex or the NADH
dehydrogenase‐like (NDH) complex, thus driving cyclic electron
transfer (CET) around PSI and sustaining ATP synthesis (Ma
et al., 2021). The membrane‐embedded NDH‐1 complex not
only reduces the PQ pool for CET with electrons deriving from
NADPH via FNR and Fd (Mulo and Medina, 2017) but also
transfers protons into the thylakoid lumen for activation of the
ATP synthase (Peltier et al., 2016). NDH‐1 is also proposed to
be involved in a respiratory pathway inside the chloroplasts,
called chlororespiration, which includes reoxidation of the PQ
pool and reduction of oxygen to water by the membrane protein
Plastid Terminal Oxidase (PTOX) as an alternative electron sink.
PTOX was hypothesized to have a dual function, depending on
the prevalent light intensity and thus the redox state of the PQ
pool/lumen pH (Yu et al., 2014; Feilke et al., 2016; Krieger‐
Liszkay and Feilke, 2016). Current models suggest that under
high light conditions, PTOX associates with the thylakoid
membrane and is accessible for its substrate plastoquinol
(PQH2), hence oxidizing the over‐reduced PQ pool, using
oxygen as an electron acceptor. However, in a side reaction,
superoxide and hydroxyl radicals (ROS) are produced which
could lead to photoinhibition of the photosystems if not scav-
enged properly by other antioxidant systems (Sun et al., 2017).
Under nonsaturating light, on the other hand, it was demon-
strated that PTOX can theoretically act as an extra electron sink
and keep the PQ pool more oxidized and protected from
photoinhibition, although its expression levels are extremely low
under this condition, avoiding competition with LET (Feilke
et al., 2016). However, this “antioxidant” feature is important
under dynamic light conditions (Nawrocki et al., 2019b). In case
superoxide is still produced at PSI, it is rapidly reduced to water
by the enzymes superoxide dismutase and ascorbate perox-
idase (Mehler reaction; Mehler, 1951). This reaction is part of the
water–water cycle, referring to electron flow from the splitting of
water at PSII to the generation of water at PSI, and acts as an
alternative electron sink to increase the ATP:NADPH ratio under
Figure 4. Arrangements of photosystems with light‐harvesting complexes (LHC) in higher plants and cyanobacteria in the thylakoid
membranes
Top views of the pigment–protein photosystem II (PSII)‐LHCII (A) and PSI‐LHCI (B) supercomplex core components in higher plants (recreated from Protein
Data Bank (PDB) entries 5MDX and 2WSC). The dimeric PSII core consists of the reaction center (RC) proteins D1 and D2 and the core proteins CP43 and
CP47. The minor antennae CP29/CP24 (heterodimer) and CP26 connect moderately and strongly bound M‐ and S‐LHCII trimers to the PSII RC via CP47
and CP43, respectively. Loosely bound L‐LHCII trimers are often detached from the supercomplex. In monomeric PSI, the RC is surrounded by LhcA1‐4 in
a fan‐like fashion, with LhcA1 and A4 and LhcA2 and A3 forming heterodimers. In contrast, cyanobacterial light‐harvesting antennae are not embedded
within the thylakoid membrane but are attached to the core proteins on the stromal surface. These large pigment‐protein complexes are called phyco-
bilisomes (PBS) and come in different shapes. Pentacylindrical PBS are predominantly present in filamentous cyanobacteria and consist of five allophy-
cocyanin (APC) core cylinders (red) from which eight phycoerythrocyanin (PEC; blue)/phycocyanin (PHC; green) rods radiate (C). The colors represent the
wavelengths the different PBS discs absorb upon binding phycocyanobilin pigments, inducing energy transfer from PEC→PHC→APC→PSII RC. Unicellular
cyanobacteria mostly contain tricylindrical PBS with three APC core cylinders and up to six rods (D). The structures were generated from PDB entries 7EYD
(Anabaena sp. PCC 7120) and 7EXT (Synechococcus sp. PCC 7002), respectively. (E) Phylogenetic tree of Arabidopsis thaliana LHC family proteins
generated in MEGAX64. Protein sequences were aligned by MUSCLE with the UPGMA cluster method. The tree was built using Maximum Likelihood as
statistical method, in conjunction with the bootstrap method (500 replications), the rtREV with Freqs. (+F) model, Gamma Distributed rates with Invariant
Sites (G+ I; five discrete gamma categories), partial deletion of gaps (95% cutoff) and the Nearest‐Neighbor‐Interchange heuristic model.
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stress conditions when more ATP is required (Asada, 2000).
Instead of stimulating ATP biosynthesis, the ATP:NADPH ratio
can also be adjusted by removing NADPH either through en-
hanced uptake by metabolic processes, such as fatty acid
production, or through transport into mitochondria via the ma-
late shuttle (for a recent review see Dao et al., 2021).
Evolution and structure of the LHC protein family
Throughout evolution, the main components of the photo-
synthetic machinery have remained well‐conserved in the plant
tree of life, ranging from single cell blue–green algae (called
cyanobacteria) to higher plants (Nelson and Yocum, 2006).
In Archean times probably more than 3.5 billion years ago, a
homodimeric photosystem reaction center diverged into Type II
and Type I reaction centers. Type II reaction centers then further
diversified into ancestral heterodimeric water‐splitting
and nonwater‐splitting systems, giving rise to oxygenic and
anoxygenic photosynthesis, respectively. Cyanobacteria are
the first known organisms to employ oxygenic photosynthesis,
having emerged about three billion years ago, releasing
molecular oxygen into the atmosphere as a by‐product of
splitting water molecules at PSII. Consequently, during the
“Great Oxidation Event” 2.4 billion years ago, the reduced, high
CO2 atmosphere was converted into an oxidized, low CO2
atmosphere—as we know it today—allowing the development
of all oxygen‐breathing organisms (Sánchez‐Baracaldo and
Cardona, 2020). Cyanobacteria are deemed the evolutionary
predecessors of the photosynthetic chloroplast organelles in
eukaryotes. Through an endosymbiotic event in which a
heterotrophic eukaryotic α‐proteobacterium engulfed an auto-
trophic cyanobacterium hundreds of million years ago, the
cyanobacterium integrated into the host cell and slowly
developed into the chloroplast over time, leading to the
generation of green algae and plant cells (Gould et al.,
2008; McFadden, 2014).
However, one major difference in the physiology of the
photosynthetic machinery between cyanobacteria and plants
is the evolution of the light‐harvesting antenna complexes.
While in green algae and plants, the pigment–protein an-
tennae are embedded in the thylakoid membrane adjacent to
the photosystems, in cyanobacteria, red algae and glauco-
phytes, they sit in a fan‐like fashion on top of the photo-
systems on the stromal side of the thylakoid membrane
(Figure 4C,D). These phycobilisomes (PBS) are large com-
plexes and comprise phycobiliproteins that can bind several
types of linear phycobilin pigments. The standard cyano-
bacterial PBS structure consists of allophycocyanin core
cylinders, from which rods composed of phycocyanin and
phycoerythrocyanin/phycoerythrin radiate. Depending on
which phycobiliprotein the pigments are bound to, they can
absorb light of different wavelengths. The distal part of the
rod absorbs short wavelengths in the blue–green range with a
maximum at 570 nm. The captured energy is then transferred
through the rods toward the PBS core, which absorbs longer
wavelengths of red light (maximum at 650 nm) and passes on
all energy to the PSII reaction center. Composition and length
of the rods can be adjusted according to the prevalent light
conditions (Bryant, 1982; Chang et al., 2015; Stadnichuk
et al., 2015; Tang et al., 2015; Green, 2019; Bag, 2021).
But “what happened to the phycobilisome?” (Green, 2019)
and how did the LHC protein family eventually evolve?
With the evolution of the chloroplast, many photosynthetic
genes from the ancestral cyanobacterium were transferred and
incorporated into the nucleus of the eukaryotic host cell. The
genes for the assembly of the PBS and the associated orange
carotenoid protein (OCP) for photoprotection (Muzzopappa and
Kirilovsky, 2020) were likely lost, possibly due to their large size
and high nitrogen requirement for protein assembly. Cyanobac-
teria also contain high light‐inducible proteins (HLIPs), which are
considered the evolutionary progenitors of the LHC protein
family. These HLIPs are small single‐helix transmembrane pro-
teins that bind chlorophyll a and the carotenoid β‐carotene and
function in assembly and photoprotection of PSII and chlorophyll
metabolism in cyanobacteria (Komenda and Sobotka, 2016; Ti-
biletti et al., 2018). Upon endosymbiosis of cyanobacterium and
eukaryote, HLIP genes were transferred into the nucleus of the
eukaryote and are still present in the plant genome as one‐helix
proteins (OHPs; Figure 4E). Through acquisition of additional
transmembrane domains, internal gene duplications and loss of
helices, two‐helix stress‐enhanced proteins (SEPs) and light‐
harvesting‐like (Lil) proteins, the nonpigmented four‐helix protein
PsbS, as well as the big group of three‐helix LHC proteins (in-
cluding early light‐inducible proteins [ELIPs]), respectively, de-
veloped especially in the green lineage of chlorophyta, ranging
from green algae to higher plants (Green, 2019; Bag, 2021). A
special case is the evolution of the presumably first LHC‐like
protein group of LHCSR proteins and the “younger” PsbS pro-
tein. In the case of PsbS, pH sensing and the active quenching
site are located on different proteins, whereas in LHCSR both
mechanisms are combined in one protein. While photoprotection
in green algae mainly relies on LHCSR proteins, in mosses, both
LHCSR and PsbS proteins are active. PsbS development and
relocation of the active quenching site to other protein com-
plexes was likely an adaptation process to more challenging light
conditions when plants started to conquer terrestrial land. This
would also explain why LHCSR proteins are completely absent in
land plants (Pinnola, 2019).
Furthermore, it is believed that CP29 was the first LHCII
protein to evolve due to its presence in taxonomically
diverse classes of green algae. This points to the existence of
an ancestral CP29 gene before the diversification of the
green lineage, followed by the rise of CP26 and ancestral
major antenna proteins LhcBM in green algae through gene
duplications and DNA crossovers of SEP genes. In contrast,
CP24 seems to be the latest addition to the LHC protein
family, being present in land plants only (Koziol et al., 2007).
Even though the protein sequence similarities are only 20%–
40%, the overall protein structures with three membrane‐
spanning domains and conserved chlorophyll a/b‐binding
sites are mostly uniform across the major and minor LHCII
proteins (Jansson, 1999; Ballottari et al., 2012). In addition to
chlorophylls, they also bind three to four carotenoids
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(xanthophyll pigments: lutein, violaxanthin, zeaxanthin, neo-
xanthin; and β‐carotene) to designated binding sites (Jahns
and Holzwarth, 2012).
IMPROVING CROP YIELDS
THROUGH OPTIMIZATION
OF PHOTOSYNTHETIC LIGHT
REACTIONS
Decreasing antenna size to increase biomass yield
In the previous sections, we described the functions of the
light‐harvesting antenna complexes in the photosynthetic
thylakoid membrane on the level of an individual leaf. How-
ever, what might be beneficial for an individual plant/leaf
might not necessarily be in favor for the entire population/
organism, respectively. As mentioned above, photosynthesis
has several rate‐limiting steps, which inhibit electron transfer
even when the energy input is maximized, causing photo-
damage of the photosynthetic machinery. In a typical crop
canopy, leaves at the top would catch most of the actinic
sunlight for the activation of photosynthesis, while light in-
cident on the leaves below will be largely composed of
wavelengths that are poorly absorbed by the LHC antennae
and will be too low in intensity to drive high rates of photo-
synthesis (Figure 5). Thus, in an optimal case, leaves in the
upper canopy would sacrifice maximum light absorption to
increase light penetration to leaves in the lower canopy to
sustain efficient photosynthesis throughout the plant. How-
ever, this is a difficult undertaking, both in traditional plant
breeding as well as with genetic engineering. Plant breeders
would normally select for traits that are beneficial for the in-
dividual plant, rather than for the entire population. For that
reason, plants with a light green color or upright leaves were
selected against in the past, signifying lower photosynthesis
rates and light absorption, respectively, on an individual plant
level, even though these traits could be favorable in a plant
community to allow better light distribution across the entire
canopy. On the other hand, plant populations with traits
beneficial for the community are often invaded by individual
plants with more competitive features. In terms of light in-
terception, examples could be taller plant height, more hori-
zontal leaf angles or bigger leaves (Anten and Vermeulen,
2016). These invaders then have an advantage in obtaining
resources over the plant community, resulting in poorer crop
yields at the stand level. With regard to genetic engineering, it
is possible to independently modify traits in different plant
tissues, but it is more challenging to independently optimize
traits in individual leaves, for instance. Nevertheless, scien-
tists have developed a range of novel approaches to tackle
this problem and reduce the LHC antenna systems for better
transmittance of sunlight through plant canopies (Ort et al.,
2011). Similar approaches have also been generated for
Figure 5. Light distribution across the plant canopy between wild type (A) and mutants with decreased light‐harvesting complex (LHC)
antenna sizes (B)
(A) In a dense crop canopy, light distribution across the leaves of the plant is very uneven because of shading effects from other leaves. Leaves of the upper
canopy closest to the light source absorb most of the photosynthetic active radiation (PAR) and deplete the light in the lower canopy of its PAR, resulting in
a strong decrease of photosynthesis with increasing canopy depth (see insert). (B) Introduction of mutants with a decreased cross‐section of the LHC
antennae allows PAR to travel deeper into the crop canopy, as the fractional PAR absorption of leaves closest to the light source has decreased. This
approach may marginally decrease photosynthesis of the light‐exposed layers but improves light distribution across the entire canopy, thus improving
overall photosynthesis in the otherwise shaded layers and the entire plant (see insert).
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microalgal/cyanobacterial liquid mass cultures in photo-
bioreactors, which are easier and faster to manipulate. Some
successful strategies could have the potential of being
translated from photosynthetic micro‐organisms into crop
species in the future, and are therefore briefly described
hereafter.
Microalgae and cyanobacteria have gained more and more
attention as potential production platforms for biotechnological,
pharmaceutical, and nutraceutical compounds over the past
decades due to their photosynthetic ability and have many
advantages over plant‐based bioengineering and production
(reviewed in the past 3 years by Benedetti et al., 2018; Khan
et al., 2018; Rizwan et al., 2018; Metsoviti et al., 2019; Naruka
et al., 2019; Mutanda et al., 2020; Zahra et al., 2020;
Dhandayuthapani et al., 2021; Khalifa et al., 2021). High pro-
duction costs are currently the major drawback owing to a low
light‐to‐biomass conversion rate inside the water column.
Hence, several attempts have been made in different algal and
cyanobacterial species to reduce the optical cross‐section of
LHCII antennae to allow better light penetration into the water
column of mass production bioreactors (Melis et al.,
1999; Mussgnug et al., 2007; Kosourov et al., 2011; Ort et al.,
2011; Cazzaniga et al., 2014; Kirst and Melis, 2014; De Mooij
et al., 2015, 2016; Shin et al., 2016; Dall'Osto et al., 2019; Hu
et al., 2020; Vecchi et al., 2020). In 2002, Polle and coworkers
targeted the unicellular green alga species Chlamydomonas
reinhardtii and Dunaliella salina for proof of this concept through
DNA insertional and chemical‐induced mutations (Polle et al.,
2002). Screening for mutants with reduced antenna size but
increased photosynthetic performance revealed several genes
that could be of interest for manipulation in order to maximize
algal biomass or hydrogen production. This list of candidate
genes predominantly included genes involved in the biosyn-
thesis of chlorophylls and carotenoids and mutants had about
20%–50% smaller light‐harvesting antennae, mainly affecting
PSII‐associated complexes. In a chl b‐less mutant, photo-
synthetic productivity could be increased two‐fold, although
lower maximum PSII quantum efficiencies were observed, in-
dicating photodamage to the photosystems. This can also be
detrimental when mass cultures are grown under bright sun-
light, when photoinhibition is even more pronounced and NPQ
mechanisms are induced and waste absorbed light energy.
Therefore, Perrine and coworkers constructed chlorophyllide a
oxygenase (CAO) RNA interference (RNAi) mutants in Chlamy-
domonas to obtain transgenic strains with intermediate‐sized
antenna complexes (20%–30% reduction in LHCII) instead of
chl b‐less strains with total loss of the peripheral PSII antenna
system (Perrine et al., 2012). The CAO RNAi mutants showed
wild type‐like growth behavior under low light conditions but
produced significantly more biomass (15%–35% compared to
the wild type) when exposed to high light, without being im-
paired in photoprotective mechanisms, such as state transitions
and xanthophyll‐dependent NPQ. However, one drawback is
the reduced flexibility of these mutants to adjust antenna size to
the prevailing conditions, especially outdoors, where light can
show sharp dynamic fluctuations. To target this issue, the same
authors improved their previous system by fusing the CAO
gene to a light‐responsive transcription factor‐binding site of the
LHCMB6 gene and transferring this construct into a chl b‐less
mutant background (Negi et al., 2020). With this system, the
translational repressor NAB1 will bind to the CAO gene upon
illumination and inhibit its expression, thus downregulating the
LHCII antenna size in a light‐dependent fashion. These trans-
genic Chlamydomonas strains indeed showed highly dynamic
adjustments of the antenna systems under fluctuating light
conditions, with wild type‐like PSII quantum efficiencies, slightly
lower NPQ and a three‐fold increase in biomass compared to
control strains under dynamic light. It seems that this successful
proof of concept may also have potential to improve
productivity of crop species.
In crops, modifying light‐harvesting cross‐sections has
led to a range of outcomes over the past 10 years. While
soybean mutants with reduced chlorophyll levels did not
show any increase in biomass accumulation when grown in
the field (Slattery et al., 2017), a rice mutant expressing a
maize GOLDEN2‐LIKE (GLK) transcription factor demon-
strated enhanced chlorophyll and antenna complex biosyn-
thesis, surprisingly resulting in 30%–40% more biomass and
grain yields compared to wild type plants (Li et al., 2020). GLK
proteins induce chloroplast development through regulation of
plastid and nucleus‐encoded genes. Overexpression of GLK can
even lead to the development of chloroplasts in nongreen tis-
sues, such as roots. The resulting root photosynthesis of trans-
genic lines of the model plant Arabidopsis thaliana contributed to
a small extent to overall CO2 assimilation in addition to leaf
photosynthesis (Kobayashi et al., 2013). Nevertheless, the ma-
jority of studies focused on decreasing chlorophyll contents and
antenna sizes (Song et al., 2017; Bielczynski et al., 2020) with
positive outcomes for improving crop yields (Jin et al.,
2016; Kirst et al., 2017; Friedland et al., 2019). While Jin and
colleagues knocked out a chloroplast protein that regulates
translation of chlorophyll biogenesis genes (HIGH PHOTO-
SYNTHETIC EFFICIENCY1, HPE1), Kirst and coworkers made
use of an established yellow–green line with truncated light‐
harvesting antennae in the model plant Nicotiana tabacum (to-
bacco) and observed higher biomass accumulation per unit
absorbed light. Consequently, they proposed a shift in plant
agronomy by reducing the space left between sowed plants in
the field, as a strategy to increase the biomass outcome per
field. Nevertheless, there is a fine balance between reducing the
optical cross‐section of the LHC antennae for improved yields
and preserving their functions in photoprotective mechanisms
(Wu et al., 2020). It is also important to note that manipulation of
the light‐harvesting antennae is easier to accomplish in uni-
cellular/multicellular microalgae/cyanobacteria compared to
highly differentiated, multiorgan systems, such as plants.
Improving NPQ features increases biomass
accumulation in dynamic light conditions
A complex component of photosynthesis is photoprotection
of the electron transport chain from overexcitation under high
light conditions. Photoprotective mechanisms are activated
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upon high light exposure to prevent overexcitation or dis-
sipate excess absorbed light energy as heat (NPQ). Thus, in a
quenched state, photosynthetic efficiency is downregulated
until more favorable light conditions occur. However, upon
shift from high light to low light conditions, NPQ mechanisms
relax relatively slowly compared to their rate of induction, still
inhibiting efficient photosynthesis for several minutes upon
transition to more favorable light conditions (Zhu et al., 2004).
During these minutes, valuable energy for photosynthetic
biomass production is lost, which is particularly problematic
under dynamic light conditions with short fluctuations in light
intensities, as is the case in natural sunlight on a cloudy day
for instance (Figure 6). Several studies have shown that plant
growth is significantly reduced under fluctuating light
conditions (Leakey et al., 2002; Kubásek et al., 2013
; Vialet‐Chabrand et al., 2017; Kaiser et al., 2020). Scientists
therefore aim to accelerate NPQ relaxation kinetics under
fluctuating light through modification of the molecular players
involved, in order to improve crop yields.
In an attempt to speed up NPQ relaxation kinetics in
dynamic light conditions, Kromdijk and coworkers simulta-
neously overexpressed the PsbS protein and both xanthophyll
cycle enzymes VDE and ZEP in tobacco (Kromdijk et al., 2016).
Transgenic tobacco VPZ overexpression lines exhibited no sig-
nificant differences from wild type tobacco plants when grown
under steady state light conditions. However, under fluctuating
light, VPZ lines outperformed control plants by 15%–20% in-
crease in biomass under greenhouse as well as field‐grown
conditions, owing to faster NPQ relaxation kinetics and im-
proved net CO2 assimilation. Since these NPQ components are
highly conserved in higher plants, this approach has great po-
tential of increasing future crop yields. Nevertheless, expression
of a VPZ construct in Arabidopsis did not result in the previously
observed growth advantage in tobacco lines (Garcia‐Molina and
Leister, 2020), demonstrating that different mechanisms might
be at work and that cloning vector units might need optimization
in different plant species. Furthermore, an overexpression
line of PsbS in rice did also show about 20% increase in canopy
radiation use efficiency into biomass and grain yield under
fluctuating light (Hubbart et al., 2018). Modeling and 3D
reconstruction of the rice canopy revealed that leaves in the
lower canopy have higher capacities for photoprotective NPQ
than leaves from the upper canopy (Foo et al., 2020), which may
reflect the greater occurrence of fluctuations in light due to
shading from the upper canopy and sunflecks. These results
show the importance of considering the canopy context when
trying to design strategies for photosynthetic improvement. In
contrast to overexpressing PsbS in rice, tobacco plants over-
expressing PsbS did not reveal any gain in biomass compared
to control plants when grown under controlled and field
conditions (Głowacka et al., 2018). However, these plants did
display an increased water use efficiency of 25%–33% due
to dampening of the light‐induced increase in stomatal
conductance. Nevertheless, photosynthesis in general was not
affected by PsbS overexpression. The authors proposed
that PsbS abundance might modulate the redox state of the
plastoquinone pool in the thylakoid membrane, which had been
put forward as an early signal for stomatal opening in response
to light (Busch, 2014). Whereas these studies exemplify the
importance of the PsbS protein, there might be other genetic
factors that could be utilized to adjust NPQ traits for improved
crop performance. Quantitative genetics and genome‐wide
association studies targeting natural variation in NPQ traits
have already been done in soybean, rice, and Arabidopsis,
revealing 15–33 putative new targets for manipulation of NPQ
(Jung and Niyogi, 2009; Herritt et al., 2016; Wang et al.,
2017; Rungrat et al., 2019).
A different avenue to explore could be the introduction of
a different xanthophyll cycle into future crop plants. While the
violaxanthin cycle is the predominant xanthophyll cycle in
most tested plant species, a lutein epoxide cycle works in
parallel to the violaxanthin cycle in some nonmodel plant
species (Bungard et al., 1999). Epoxidation of lutein confers
Figure 6. Activity of nonphotochemical quenching (NPQ)
mechanisms and xanthophylls under changing light conditions
and their potential for improving photosynthetic efficiency
Plants often encounter sudden changes in light intensities (sunflecks or
shading), which they quickly need to respond to and adjust their metabolic
setup in order to avoid photodamage or maintain their photosynthetic
capacity. Upon exposure to high light intensities, NPQ (black lines) com-
ponents are swiftly initiated to dissipate excess excitation energy as heat
and prevent photoinhibition of the photosynthetic machinery. Acidification
of the thylakoid lumen initiates the fastest NPQ component qE (energy‐
dependent quenching) within seconds to minutes, which is subsequently
further enhanced through activation of the xanthophyll cycle, that is, the
conversion of violaxanthin (V) into photoprotective zeaxanthin (Z) via an-
theraxanthin (A). Simultaneously, NPQ inhibits photosynthetic efficiency
(red lines), which drops to very low levels under high light conditions. Upon
the shift to low light or dark conditions, NPQ relaxes and pigment–protein
photosystem II (PSII) efficiency recovers, observable through reconversion
of zeaxanthin to violaxanthin. However, full relaxation of NPQ after high
light stress is a rather slow process (30–60min or longer), during which
photosynthetic capacity is still inhibited to some extent under otherwise
optimal conditions, thereby possibly losing time for biomass production.
By overexpressing the lumenal pH sensor protein PsbS and the
xanthophyll‐converting enzymes in tobacco (dashed lines), transgenic VPZ
plants displayed faster NPQ relaxation under changing light conditions
and thus faster recovery of photosynthesis, which resulted in higher bio-
mass accumulation compared to control plants (Kromdijk et al., 2016).
Graphs displayed here are schematic representations.
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higher light‐harvesting efficiency and has been successfully
engineered into Arabidopsis lines through expression of a
ZEP gene from Nannochloropsis oceanica (Leonelli et al.,
2017). Transgenic plants only expressing the lutein epoxide
cycle displayed significantly higher PSII maximum quantum
efficiencies and similar NPQ kinetics compared to plants
employing the violaxanthin cycle, pointing at the potential of
this alternative xanthophyll cycle for future studies.
All these different approaches have one thing in
common—they require acidification of the thylakoid lumen,
that is, a low pH through accumulation of protons. Lumen
acidification is also accompanied by fluxes of counter‐ions
over the thylakoid membrane, which help to maintain the
balance between the ΔpH and the electric field
(ΔѰ) component of the pmf. Influx of potassium ions (K+) into
the lumen upon the shift from light to dark for instance in-
creases ΔѰ, thereby allowing a decrease in ΔpH and the
relaxation of the qE component of NPQ without com-
promising overall pmf to maintain ATP synthase activity (si-
mulated by Davis et al., 2017). Therefore, counter‐ion fluxes
could possibly be a novel target for modification of NPQ
mechanisms (Davis et al., 2017). Indeed, Armbruster and
coworkers demonstrated the significance of ion fluxes in
adaptation to fluctuating light conditions by the action of the
K+/H+ exchange antiporter KEA3, the protein family of which
is important for pH and osmoregulation inside the chloroplast
(Armbruster et al., 2014, 2016; Kunz et al., 2014; Correa
Galvis et al., 2020; Li et al., 2021). In higher plants, there are
three splice forms of KEA3, each of which has a distinct
function, but generally they export protons from the lumen
with concurrent import of potassium ions. Knockout of the
KEA3 gene in Arabidopsis resulted in a specific NPQ phe-
notype with higher NPQ levels upon the shift from dark to low
light and slower NPQ relaxation from high to low light, which
was attributed to the qE component of NPQ (Armbruster
et al., 2014). Independent overexpression of the three KEA3
splice forms in the KEA3 knockout background both in Ara-
bidopsis and transiently in Nicotiana benthamiana showed
the specific contribution of each isoform to this NPQ phe-
notype. KEA3.2 is the most abundant isoform and its over-
expression led to lower NPQ levels than the wild type in the
initial induction response in the transition from dark to low
light, but not at high light, reflecting the opposite trends in
NPQ kinetics of the KEA3 knockout mutant. Thus, the
oeKEA3.2 line also displayed significantly faster NPQ relax-
ation under fluctuating light through enhanced export of
protons from the lumen. Overexpression of KEA3.3 had a
similar low NPQ response to oeKEA3.2 in the shift from dark
to low light. In contrast to oeKEA3.2 though, oeKEA3.3 also
had a significantly lower NPQ amplitude in the transition to
high light conditions, whereas oeKEA3.2 showed almost wild
type levels during NPQ induction in high light despite its
overexpression. These results indicate that KEA3.3 is more
active than KEA3.2 in exporting protons from the lumen
under high light stress, thus reducing and regulating overall
NPQ levels. KEA3.2 and KEA3.3 splice forms distinguish by
the presence of a KTN domain in KEA3.2, which is responsive
to changes in NADH/NAD+ or ATP/ADP ratios or could pos-
sibly be regulated by the redox state of the plastoquinone
pool (Armbruster et al., 2016). Overexpression of KEA3.1 did
not show any special NPQ phenotype compared to the other
isoforms and only slightly ameliorated the low/slow NPQ re-
sponse of the KEA3 knockout mutant, which exhibited a
significantly slower growth rate when grown under fluctuating
light. Surprisingly, no significant growth advantage of the
overexpression lines could be detected, although oeKEA3.2
showed trends of enhanced growth. Further analyses re-
vealed that KEA3 activity requires more fine‐tuning in order to
optimize photosynthesis (Wang et al., 2017) and that com-
binations with other players involved in the generation of a
ΔpH gradient over the thylakoid lumen (NDH complex in
cyclic electron flow) can amplify the KEA3 phenotype (Basso
et al., 2020).
Many of the fore‐mentioned studies have not investigated
the effects of ROS accumulation in the transgenic lines, but the
manipulations are likely to have an impact on photo‐oxidative
stress alleviation in addition to improved photosynthetic effi-
ciencies (as suggested by Davis et al., 2017). Photo‐oxidative
stress is established through the generation of the ROS singlet
oxygen and superoxide, predominantly arising from PSII and
PSI, respectively, through overreduction of the electron
transfer chain. ROSmainly damage the core protein D1 of PSII,
thereby downregulating photosynthesis under long‐term
unfavorable conditions (photoinhibition). PSII employs an ef-
fective D1 repair cycle with de novo protein biosynthesis;
however, this is a rather energy cost‐intensive process (Baena‐
González and Aro, 2002). In attempts to engineer tobacco
plants with enhanced resistance to drought stress, Almoguera
and colleagues overexpressed the heat‐shock transcription
factor A9 (HSFA9). This transcription factor was presumed to
activate the expression of small heat‐shock proteins in chlor-
oplasts and indeed conferred improved drought and oxidative
stress tolerance as visible from sustained D1 protein levels
after withholding of water (Almoguera et al., 2012). Similar re-
sults were achieved with an alternative approach in tobacco
overexpressing a plastid‐encoded full‐length PsbA gene from
maize under the control of the 35S promoter. These mutant
lines had increased levels of the D1 protein and showed en-
hanced drought tolerance under stress conditions but wild
type‐like growth phenotypes under optimal conditions (Huo
et al., 2016). Following up on these studies, Chen and
coworkers then combined and optimized both approaches and
engineered a heat responsive PsbA construct expressed in the
nucleus (Chen et al., 2020). The native PsbA gene is encoded
in the chloroplast genome which is closer to the site of the
PSII/D1 repair cycle for de novo synthesis upon recovery from
photoinhibition. However, ROS production inside the chlor-
oplast can strongly inhibit the translation of the PsbA mes-
senger RNA (mRNA) into the D1 protein (Nishiyama et al.,
2001, 2004). By transferring PsbA expression to the cell nu-
cleus, D1 de novo synthesis could take place in the cytosol
instead of the chloroplast. The mature protein was targeted to
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the chloroplast with the help of a chloroplast transit peptide
(from RbcS) and remarkably, was able to replace degraded D1
protein in the PSII complex. Placing PsbA gene expression
under the control of a heat responsive promoter from the
HSFA2 (heat‐shock transcription factor A2) gene, enhanced
gene expression upon exposure to increased temperatures.
This presumably explained the enhanced heat stress tolerance
in transgenic lines of Arabidopsis, tobacco, and rice, all of
which also had significantly enhanced growth, biomass and
grain yield both under nonstressed conditions and in field trials.
Altogether, these results prove that photosynthesis can effi-
ciently be upregulated through mitigation of photo‐oxidative
stress and photoinhibition, leading to yield benefits in monocot
as well as dicot plant species.
Increasing electron transport capacity
Photosynthesis is composed of a series of electron transfer
steps in the light reactions and enzymatic reactions in the
Calvin–Benson–Bassham cycle. One of the major protein
complexes within the thylakoid membrane besides the two
photosystems is the cyt b6f complex, which is reduced and
oxidized by the mobile electron carriers plastoquinol and
plastocyanin, respectively, in LET between PSII and PSI.
Via the Q‐cycle, the cyt b6f complex also transfers protons
across the thylakoid membrane into the lumen in addition
to its electron transfer function, thus contributing to the
generation of a lumenal ΔpH gradient that fuels ATP synthase
activity and the initiation of NPQ upon illumination. Electron
flow through cyt b6f has long been proposed to be the key
rate‐limiting step of the photosynthetic light reactions, since
mutants of several plant species with inhibited cyt b6f ex-
pression showed downregulated photosynthetic capacity
(Holloway et al., 1983; Price et al., 1995, 1998; Anderson
et al., 1997; Ruuska et al., 2000; Yamori et al.,
2011, 2016; Tikhonov, 2014). One of the eight cyt b6f
subunits is the Rieske‐FeS protein, which is encoded in the
nucleus and the expression of which determines the accu-
mulation of the entire cyt b6f complex (Anderson et al.,
1997; Price et al., 1998). Overexpressing this subunit both in
a C3 (Arabidopsis, Simkin et al., 2017b) and a C4 (Setaria
viridis, Ermakova et al., 2019) plant species gave rise to in-
creased abundance of the cyt b6f complex and enhanced
photosynthetic performance. In both studies, transgenic
overexpression plants had higher PSII quantum efficiencies,
lower NPQ values and higher CO2 assimilation rates than
control plants. In contrast to Arabidopsis transgenic plants,
Setaria overexpression lines were not reported to display any
growth advantage over wild type plants, despite the 10%
increase in CO2 assimilation. Arabidopsis overexpression
lines, on the other hand, had 30%–70% more biomass and
up to 50% greater seed yield compared to control plants. In both
species, nevertheless, the abundance of the cyt b6f complex and
the rate of CO2 fixation seem to be correlated through control of
the electron transfer rate by cyt b6f. It will be interesting to see
whether this crop improvement strategy could have similar out-
comes in other C3 and C4 crop species in the future.
The next step within photosynthetic light reactions
downstream of the cyt b6f complex involves electron transfer
through the thylakoid lumen toward PSI via the soluble pro-
tein plastocyanin, which is encoded by two genes (PetE) in
higher plant genomes. This electron carrier has been shown
to be essential for light‐dependent electron transfer as a
double knockout mutant of both PetE genes is only viable
when grown heterotrophically on media containing sucrose,
but not when grown on soil only (Weigel et al., 2003).
Therefore, plastocyanin‐mediated electron transfer might be
a rate‐limiting step in higher plant photosynthesis as well
(Burkey, 1994; Burkey et al., 1996; Schöttler et al., 2004
; Finazzi et al., 2005; Höhner et al., 2020), although even low
levels of this protein seem capable of efficiently sustaining
photosynthetic light reactions (Abdel‐Ghany, 2009; Pesaresi
et al., 2009). Attempts have been made to overexpress
plastocyanin genes in Arabidopsis, which indeed resulted in
enhanced biomass production of up to 1.6‐fold compared to
the wild type, even though photosynthetic parameters were
similar to wild type levels and no improvement of photo-
synthetic capacity could be observed (Pesaresi et al., 2009).
This phenomenon could possibly be explained with the newly
discovered role of plant plastocyanins in oxidative stress
tolerance (Zhou et al., 2018). Plastocyanins are copper‐
binding proteins and expression of the PetE genes is highly
dependent on copper availability. While PetE2 is the pre-
dominant plastocyanin expressed under nonstress and
copper‐enriched conditions and acts as a buffer in copper
homeostasis, PetE1 expression increases upon copper
starvation and functionally replaces PetE2 (Abdel‐Ghany,
2009). Under stress conditions, copper is released into the
chloroplast via two P‐type ATPases (Abdel‐Ghany et al.,
2005) and reacts with H2O2 in a Fenton reaction, generating
the highly reactive ROS hydroxyl radical (Sutton and
Winterbourn, 1989). Through introduction of the PetE2 gene
from the halophytic plant Suaeda salsa (Song and Wang,
2014) into Arabidopsis, a plastocyanin with a greater copper‐
binding capacity was able to confer a greater tolerance of
transgenic Arabidopsis lines to oxidative stress than the
original Arabidopsis plastocyanins (Zhou et al., 2018). This
resulted in stress‐tolerant Arabidopsis plants with a fresh
weight three to four times higher than that of wild type plants
grown under ROS producing stress conditions. It is, there-
fore, of high importance to explore natural variation of protein
features, especially in plant species living in extreme envi-
ronments and to exploit their potential of being translated
into agricultural crop species. In addition, it may also be
important to look further than just proteins for the regulation
of photosynthetic processes. Other regulatory elements,
such as microRNAs, for post‐transcriptional gene expression
regulation have gained more and more interest in the past
20 years (Meyers and Axtell, 2019; Wang et al., 2019). So far,
no microRNA has been discovered that directly controls
plastocyanin gene expression. However, through an indirect
mechanism, one of the microRNAs with high conservation in
plants, miR408, was found to impact copper levels inside
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the chloroplast through gene regulation of two copper trans-
porters in the chloroplast envelope and thylakoid membranes.
Overexpression of miR408, hence, led to increased expression
of plastocyanin as well as other photosynthetic genes, resulting
in enhanced biomass accumulation of 10%–20% in Arabi-
dopsis, tobacco, and rice plants (Pan et al., 2018). Interestingly,
this approach not only yielded improved vegetative plant
growth but also boosted seed and grain size and weight even
under field‐grown conditions. Moreover, miR408 seems to be
highly conserved in eudicot and monocot plant species, making
it a promising target for improved crop growth.
The last step of LET encompasses the protein FNR, which
accepts electrons from Fd and subsequently reduces NADP+
to NADPH. Efforts have been made to overexpress FNR in
tobacco, but no growth phenotype could be observed
despite an increase in oxidative stress tolerance (Rodriguez
et al., 2007). Knockdown of FNR, on the other hand, drastically
decreased the mutants' photosynthetic capacity and made it
highly susceptible to oxidative stress (Lintala et al., 2012).
However, recently it has been proposed that the interaction
between FNR and the Thylakoid RhOdanase‐Like protein
(TROL), the protein that putatively binds FNR to the thylakoid
membrane, could have potential for crop improvement
(Fulgosi and Vojta, 2020). TROL is a membrane‐spanning protein
close to PSI in the grana margins, with a rhodanase‐like domain
(RHO) exposed to the thylakoid lumen. Here, the RHO domain
may be involved in sensing redox signals, upon which FNR‐
binding on the stromal side to the membrane recruiting motif
(MRM) of TROL is adjusted, possibly in a light‐ and pH‐
dependent manner. It is postulated that FNR is membrane‐
bound in the dark, reversing its function and providing reduced
Fd to a number of metabolic pathways, including oxidative stress
tolerance. Upon light exposure, FNR is released into the stroma,
only then being active in LET toward NADPH regeneration.
Hence, TROL could potentially be a target for the switch in FNR
action mode, either through modifications of the redox sensor
domain RHO or the FNR‐binding domain MRM.
Instead of forwarding electrons to FNR, Fd can also
reduce components of AET routes to stimulate ATP synthesis
under stress conditions. However, the exact mechanisms are
not yet clear and contrasting views on the function of these
AET complexes as alternative electron acceptors down-
stream of PSI have recently been brought forward (Nawrocki
et al., 2019a; Buchert et al., 2020; Rantala et al., 2020; Zhou
et al., 2020; Rühle et al., 2021; Wu et al., 2021). Nevertheless,
it is well‐accepted that the energy balance is of high im-
portance when it comes to altering metabolic processes
(Kramer and Evans, 2010). Reducing the levels of PGR5
protein seems to negatively impact plant growth to some
extent under fluctuating light conditions (Munekage et al.,
2008; Nishikawa et al., 2012), whereas in Chlamydomonas,
deletion of this protein promotes biotechnological hydrogen
production due to rerouting of electrons toward the hydro-
genase HydA upon stress induction (Steinbeck et al.,
2015; Nagy et al., 2021). Relative to C3 species, C4 plant
species rely more strongly on CET around PSI in bundle
sheath cells in order to provide extra ATP to fuel the carbon
concentrating mechanism. However, although over-
expression of PGR5 in Flaveria bidentis led to alleviation of
PSI acceptor side limitation, it did not result in enhanced
CO2 fixation (Munekage et al., 2010; Tazoe et al., 2020).
In contrast, overexpression of PGR5 resulted in increased
growth rates in diatoms under fluctuating light (Zhou et al.,
2021) and enhanced high light and drought stress in Arabi-
dopsis (Long et al., 2008). In addition, PTOX is also important
as an alternative electron sink and for chloroplast biogenesis
and carotenoid biosynthesis during early leaf development,
and induction of its expression under stress conditions
(summarized in Sun and Wen, 2011 and Johnson and
Stepien, 2016; Ivanov et al., 2012; Li et al., 2016a;
Ghotbi‐Ravandi et al., 2019) led to the suggestion that it
could be a suitable target for enhancing stress tolerance in
plants (Johnson and Stepien, 2016). However, initial experi-
ments overexpressing PTOX in Arabidopsis and tobacco did
not result in enhanced tolerance to high light stress but in-
stead made plants more photosensitive (Joët et al.,
2002; Rosso et al., 2006; Heyno et al., 2009; Ahmad et al.,
2012, 2020). Nevertheless, overexpression of PTOX did
provide an advantage when exposed to salt stress. This ef-
fect seemed to rely on a translocation of PTOX from the
stroma lamellae to the appressed grana stacks in salt‐
tolerant plants (Stepien and Johnson, 2018; Ahmad et al.,
2020). This correlation between PTOX expression and salt
stress tolerance was also recently confirmed in a halophyte
C4 plant species (Essemine et al., 2020).
Instead of indirectly upregulating the production of ATP
through upregulation of CET routes, it is conceivable to di-
rectly enhance chloroplast ATP synthase activity (Cardona
et al., 2018; Davis and Kramer, 2020). The ATP synthase is
composed of two rotary motor complexes (CF0/CF1) which
are connected by two flexible stalks (Kühlbrandt, 2019). The
CF0 complex is embedded within the thylakoid membrane
and consists of a ring of several c‐subunits, the numbers
of which are organism/species‐dependent but remain
constant under different conditions (between eight and 17
c‐subunits; Davis and Kramer, 2020). Each c‐subunit binds
one proton from the thylakoid lumen, fueling the CF0 motor
and subsequently the CF1 motor on the stromal side of the
membrane, thus promoting the production of three ATP
molecules per 360° rotation. Davis and Kramer (2020) recently
proposed a theoretical model from kinetic simulations
of photosynthetic reactions that considers the size of the
c‐ring/number of c‐subunits per ring and therefore the ratio of
required protons per generated ATP. A lower ratio (a smaller
c‐ring size) would theoretically result in higher photosynthetic
energy conversion rates. However, the simulations predicted
that a smaller ring size would also lead to a higher ΔpH across
the membrane, thus activating photoprotective NPQ mecha-
nisms and limiting photosynthetic efficiencies even under dark
conditions. Evolution has therefore favored a bigger ring
size instead, in order to avoid photodamage at the cost
of conversion efficiencies. Nevertheless, the concept of
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optimizing the H+/ATP ratio (Pogoryelov et al., 2012) could
provide a novel avenue for improving photosynthesis,
considering further adjustments to the photosynthetic machi-
nery. In a different approach, mutant lines with point mutations
in ATP synthase genes were analyzed in different organisms.
In the cyanobacterium Synechococcus elongatus sp. PCC
7942, a single nucleotide polymorphism (SNP) was identified
in the atpA gene, which enhanced ATP synthase contents and
activity, conferring improved photosynthetic efficiency as well
as carbon fixation rate, especially under stress conditions (Lou
et al., 2018). Certain point mutations in the AtpB gene of the
CF1 complex, on the other hand, appear to be deleterious for
the assembly of the entire complex and normal plant growth,
so that mutants spontaneously reverted to the wild type gene
sequence (Robertson et al., 1989; Malinova et al., 2021).
Additionally, a SNP of a threonine residue in the β‐subunit of
CF1 (T86A in AtpB) was identified in cold‐tolerant cucumber
species when the gene sequences were compared to
cold‐susceptible species. Threonine residues are often subject
to post‐translational modification with regulatory phospho
groups. Lack of a phosphorylation site in AtpB due to this SNP
could possibly change its mode of action and therefore confer
improved tolerance to cold stress (Oravec and Havey, 2021).
Furthermore, an Arabidopsis mutant line with altered ATP
synthase regulation was isolated from an ethyl methanesul-
fonate library, revealing a point mutation in the γ1‐subunit of
the central stalk (Wu et al., 2007; Kanazawa et al., 2017). This
mutation resulted in about 50% loss of ATP synthase protein
content without compromising its overall activity under low light
conditions when compared to the wild type, indicating a higher
activity of the remaining complexes in the mutant. However,
when exposed to stressful conditions, such as low CO2 and
fluctuating light, the mutant was much more susceptible to
photoinhibition. The authors concluded that this particular
protein residue is involved in the stress‐related downregulation
of the ATP synthase activity, rendering a fraction of the ATP
synthase pool inactive to prevent overreduction of the electron
chain (Kanazawa et al., 2017). These results show that it is not
sufficient to simply aim for constantly enhanced ATP synthase
activity, but that it is necessary to consider its regulatory
mechanisms under suboptimal conditions as well to maintain
the plant's capacity for the induction of NPQ and a healthy ATP
to NADPH ratio. Such control mechanisms do not only act on
the membrane‐bound CF0 complex through the pmf but are
also administered through thiol‐based redox regulation of
cysteine residues on the γ‐subunit of the CF1motor (Yang et al.,
2020; Buchert et al., 2021).
Thioredoxins are proteins that reduce disulphide bonds
of cysteines in a light‐dependent manner, thus regulating
protein activity (Nikkanen and Rintamäki, 2019). A chloroplastic
NADPH‐dependent thioredoxin reductase C (NTRC) is known to
interact with the ATP synthase γ‐subunit (Nikkanen et al., 2016).
Overexpression of NTRC in Arabidopsis resulted in significantly
enhanced biomass accumulation, starch production, photo-
synthetic efficiency, NDH‐dependent CET and photo‐oxidative,
drought and heat stress tolerance compared to the wild type,
especially under low light conditions, which was mainly attrib-
uted to lower acceptor side limitation of PSI (Chae et al.,
2013; Toivola et al., 2013; Nikkanen et al., 2016, 2018; Kim et al.,
2017). Interestingly, the overexpression mutant also had lower
NPQ under light‐limiting conditions up to about 500 µmol pho-
tons/m2/sec and displayed much faster NPQ relaxation upon
high to low light transitions under fluctuating light conditions
(Guinea Diaz et al., 2020). However, NTRC overexpression in
tobacco resulted in a slight growth retardation during early plant
development when compared to the wild type despite higher
leaf starch content (Ancín et al., 2019). The increased starch
content was suggested to derive from decreased starch turnover
during the night rather than enhanced starch biosynthesis during
the day. A connection between redox regulation and starch
metabolism is present in a range of plant species and organs
(Sanz‐Barrio et al., 2013; Hou et al., 2019) and might have
potential for future bioengineering of starchy crops (Nikkanen
et al., 2017).
Translating strategies from lower plants/microalgae/
cyanobacteria into higher plants
Throughout evolution, oxygenic photosynthesis has developed
in cyanobacteria first and subsequently in microalgae
and plants upon endosymbiotic events with heterotrophic
eukaryotes. Different photosynthetic organisms had to adjust to
different environmental conditions, depending on the prevalent
light and CO2 levels. Many years of basic research have
revealed different traits and mechanisms and variations in
protein complexes between aquatic and terrestrial species that
have been optimized to adjust to certain stress conditions. It is,
therefore, of high interest to translate this basic knowledge of
possibly more effective ancestral proteins and potentially ad-
vantageous mechanisms from lower organisms into higher
plants to ultimately improve photosynthetic performance in
crops. Successful examples of this concept include the ex-
pression of cyanobacterial/algal Calvin cycle enzymes in several
plant and crop species (recently reviewed in Simkin et al., 2019).
One success story has been the introduction of the algal
cytochrome c6 (cyt c6) protein into Arabidopsis and tobacco,
which improved photosynthetic electron transfer and biomass
accumulation even under field conditions (Chida et al.,
2007; Yadav et al., 2018; López‐Calcagno et al., 2020). This
soluble protein is present in many cyanobacteria and green algae
species and is located in the thylakoid lumen where it
shuttles electrons between the cyt b6f complex and PSI for
light‐dependent LET of photosynthesis, in the same fashion
plastocyanin operates, as well as between cyt b6f and terminal
oxidases for respiration in cyanobacteria (Torrado et al.,
2019; Viola et al., 2021). Cyt c6 and plastocyanin expression
levels are dependent on iron and copper availability, respectively,
(García‐Cañas et al., 2021) as plastocyanin binds copper,
whereas cyt c6 is an iron‐binding protein that seems to have
been lost in green plants after the Great Oxidation Event (recently
reviewed in Castell et al., 2021b and Slater et al., 2021). When the
atmosphere became enriched in oxygen, which readily reacts
with iron, the level of available iron ions as co‐factors for cyt c6
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was limited, thus promoting the activity of plastocyanin in green
plants instead. In cyanobacteria, there are two more cyt c6 gene
homologs present, which were proposed to have arisen from cyt
c6 gene duplications and were annotated as cyt c6B. Interest-
ingly, green plants lost the cyt c6 and cyt c6B genes but instead
contain a gene homolog, cyt c6A, which likely evolved from cyt
c6B through insertion of a loop insertion peptide (Slater et al.,
2021). However, both cyt c6A and c6B proteins show much
lower redox midpoint potentials compared to cyt c6 and are,
therefore, not likely to contribute to electron transfer in the thy-
lakoid lumen, in contrast to cyt c6 (Molina‐Heredia et al.,
2003; Bialek et al., 2014). It has been demonstrated that cyt c6
proteins from different algae/seaweed species are suitable for
introduction into plant model species to effectively perform
electron transfer, having been selected based on a similar redox
midpoint potential to plant plastocyanins. While Chida and
coworkers inserted a cyt c6 gene from the red alga Porphyra
yezoensis into Arabidopsis (Chida et al., 2007), Yadav and
coworkers utilized a cyt c6 gene from the green macroalga Ulva
fasciata (sea lettuce) and introduced it into tobacco (Yadav et al.,
2018). In both cases, the algal genes were under the control
of the constitutive cauliflower mosaic virus 35S (CaMV35S)
promoter and were fused to a plant species‐specific PetE transit
peptide for correct localization of the cyt c6 protein into the
chloroplast thylakoid lumen. Both studies reported enhanced
growth phenotypes during the first 8 weeks of plant growth, in
accordance with increased chlorophyll and photosynthetic me-
tabolite contents, although other photosynthetic parameters
were only slightly improved. An even more advanced approach
was recently undertaken by López‐Calcagno and coworkers by
combining enhancements of LET and carbon fixation in two
tobacco cultivars through addition of an algal cyt c6 gene
from Porphyra umbilicalis and the bifunctional cyanobacterial
FBP/SBPase from Synechocystis sp. PCC 7942 or SBPase gene
from higher plants, respectively (López‐Calcagno et al., 2020).
While the single mutants had slightly increased photosynthetic
rates, the double mutants showed significantly enhanced CO2
assimilation rates (up to 15% more than control plants) and PSII
operational efficiencies. Consequently, single mutants had 9%
–44% and double mutants had 32%–52% more biomass than
control plants when grown in a controlled environment in a
glasshouse. Field experiments with these mutants, on the other
hand, showed much more variation in terms of biomass gains.
During a small field trial in 2016, leaf material from single mutants
was harvested before the flowering stage and again displayed a
20%–44% increase in biomass accumulation. However, in the
following field season only a small growth advantage of
the double mutants compared to control plants was visible when
plant material was harvested after the onset of flowering.
Surprisingly, photosynthetic parameters were not significantly
different from control plants, although an improved intrinsic water
use efficiency could be measured. Nevertheless, these mutant
constructs targeting both the light reactions as well as carbon
fixation seem to have great potential for improving crop yields,
especially for plant species with short generation times.
In addition, it was also demonstrated that introduction of a
plastocyanin gene from Chlamydomonas into the diatom
Phaeodactylum tricornutum, which normally only contains cyt
c6, can enhance biomass production under iron‐deficient con-
ditions by 60% when compared to the wild type (Castell et al.,
2021a). All these studies show that it would be indeed beneficial
for photosynthetic organisms to employ both soluble electron
carriers plastocyanin and cyt c6 since either protein can
functionally replace the other one when nutrients are short of
Figure 7. Schematic overview of light reaction components that have been targeted for bioengineering improved crops
Strategies for boosting plant photosynthesis through manipulations of light reaction components are highlighted in either red or blue in this depiction
of Figure 3. Components that have not been investigated further yet are depicted in gray. Proteins and protein complexes that are highlighted in red have
been directly overexpressed or their expression has been indirectly induced and resulted in a measurably improved phenotype compared to control plants.
Blue highlighting of protein complexes indicates downregulation of these components. Yellow–orange proteins symbolize the introduction of alternative
pathways deriving from lower plants, microalgae and cyanobacteria, including an algal zeaxanthin epoxidase (ZEP), cytochrome c6 (cyt c6), flavodoxin
(FLD), and flavodiiron proteins (FDPs). Phenotypes were either associated with higher biomass accumulation (labeled with yellow asterisks) or enhanced
abiotic stress tolerance (see also Table 1). For further explanation of the light reactions see the original Figure 3.
Optimizing light reactions to improve crop yield Journal of Integrative Plant Biology
578 February 2022 | Volume 64 | Issue 2 | 564–591 www.jipb.net
Table 1. Overview of different approaches used to improve light reactions
Strategy and gene of interest
Targeted process
or protein complex Species
Phenotype
associated with
increased
biomass
accumulation
Phenotype
associated with
enhanced
abiotic stress
tolerance References
Introduction of a maize
GOLDEN2‐LIKE (GLK)
transcription factor for
overexpression of
photosynthetic genes
LHCs Rice 30%–40% — Li et al., 2020
Knockout of HIGH
PHOTOSYNTHETIC
EFFICIENCY1 (HPE1) for
impaired chlorophyll
biogenesis
LHCs Arabidopsis Yes — Jin et al., 2016
Truncated light‐harvesting
antennae mutant
LHCs Tobacco Yes — Kirst et al., 2017
RNAi of CAO for reduced
chlorophyll b expression
LHCs Camelina sativa 40% — Friedland et al., 2019
Overexpression of PsbS, VDE
and ZEP
NPQ Tobacco 15%–20% — Kromdijk et al., 2016
Overexpression of KEA3 NPQ Arabidopsis and
tobacco
Slightly — Armbruster et al., 2016
Overexpression of HSFA9 for
D1 protection
Photoprotection Tobacco — Drought stress Almoguera et al. 2012
Overexpression of
maize PsbA
Photoprotection Tobacco — Drought stress Huo et al., 2016
Engineering a heat responsive
PsbA construct expressed
in the nucleus
Photoprotection Arabidopsis,
tobacco, rice
Yes (also under
nonstress
conditions)
Heat stress Chen et al., 2020
Overexpression of the Rieske‐
FeS protein
cyt b6f Arabidopsis, Setaria
viridis
30%–70%
(Arabidopsis)
— Simkin et al., 2017b
10 increased CO2
assimilation
rate in Setaria
Ermakova et al., 2019
Overexpression of
plastocyanin
Electron transfer Arabidopsis 1.6‐fold — Pesaresi et al., 2009
Overexpression of PetE2 from
Suaeda salsa
Electron transfer
by plastocyanin
Arabidopsis 3–4‐fold Oxidative stress Zhou et al., 2018
Overexpression of miR408 for
enhanced copper uptake
into the chloroplast
Electron transfer
by plastocyanin
Arabidopsis,
tobacco, rice
10%–20% Pan et al., 2018
Overexpression of FNR Electron transfer Tobacco — Oxidative stress Rodriguez et al., 2007
Overexpression of
(algal) PTOX
AET Arabidopsis,
Eutrema
salsugineum,
tobacco
— Salt stress Stepien and Johnson
2018; Ahmad
et al., 2020
SNPs in atpA for enhanced
ATP synthase levels
ATP synthesis Synechococcus
elongatus sp.
PCC 7942
— Heat stress Lou et al., 2018
SNPs in the β‐subunit of CF1 ATP synthesis Cucumber — Cold stress Oravec and Havey 2021
Overexpression of NTRC Redox regulation Arabidopsis,
tobacco
Yes Oxidative/
drought/heat
stress
Chae et al., 2013;
Toivola et al., 2013;
Nikkanen et al.,
(2016, 2018; Kim et al.,
2017; Ancín et al. 2019
Overexpression of algal cyt c6 Electron transfer Arabidopsis,
tobacco
Up to 50% — Chida et al., 2007;
Yadav et al.,
2018; López‐Calcagno
Continued
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either iron or copper ions, respectively, leaving room for
biotechnological improvements of crop yields.
A similar story to plastocyanin and cyt c6 can be told
about the Fd and flavodoxin proteins in plants and cyano-
bacteria, respectively. Fd is a conserved FeS electron carrier
in cyanobacteria and plants, whereas flavodoxin binds flavin
mononucleotide as cofactor and was lost from plants and
green algae during the course of evolution due to its
functional redundancy. In contrast to plastocyanin/cyt c6,
no definite answer has been found yet as to why the
iron‐containing Fd has survived the Great Oxidation Event
and has been maintained in plants, where flavodoxin has not
(Pierella Karlusich et al., 2014). In the past 15 years, many
studies on the expression of a cyanobacterial flavodoxin in
plants have been published. Most of these studies reported
on enhanced tolerance of transgenic plants to different en-
vironmental stresses, particularly iron deficiency and oxida-
tive stress, under which Fd levels would normally decrease
but can now be compensated for by flavodoxin expression
(Tognetti et al., 2006, 2007a, 2007b; Zurbriggen et al.,
2008; Shvaleva et al., 2009; Coba de la Peña et al.,
2010; Ceccoli et al., 2012; Lodeyro et al., 2012; Gharechahi
et al., 2015; Mayta et al., 2018; Gómez et al., 2020; Niazian
et al., 2021). It was also shown that flavodoxin could func-
tionally replace Fd in plants (Blanco et al., 2011) and additional
expression of a cyanobacterial FNR could increase oxidative
stress tolerance even more (Giró et al., 2011). Flavodoxin ex-
pression is not always beneficial and may lead to stunted plant
growth (Li et al., 2016b; Mayta et al., 2019) although detrimental
effects on yield were offset by a higher harvest index compared
to wild type plants (Mayta et al., 2019). Overall, flavodoxin
expression in agricultural crops seems to have great potential to
enhance abiotic stress tolerance.
Another class of photosynthetic flavoproteins that dis-
appeared in flowering plants (angiosperms) throughout evo-
lution are flavodiiron proteins (FDPs). FDPs serve as photo-
protective excess electron valves in the so‐called “Mehler‐
like reaction” or water–water cycle of photosynthesis (Allah-
verdiyeva et al., 2015; Ilík et al., 2017; Alboresi et al., 2019)
across a large part of the green lineages from cyanobacteria
up to gymnosperms. In these organisms, electron transfer on
the acceptor side of PSI, initiated by the splitting of water at
PSII, can switch from FNR and the regeneration of NADPH to
FDPs in an alternative pathway under stress conditions.
When the electron transfer chain becomes over‐reduced,
FDPs help to release excess electron pressure downstream
of PSI by reducing molecular oxygen to water, thereby
closing the water–water cycle and preventing the generation
of the highly reactive ROS superoxide for photoprotection of
PSI. In cyanobacteria, FDPs are divided into two clusters and
either work as homodimers (Mustila et al., 2016) or hetero-
dimers composed of one FDP of each cluster (Santana‐
Sanchez et al., 2019). The heterodimer Flv1/3 is conserved in
all cyanobacteria and is responsible for oxygen photo-
reduction downstream of PSI in a Mehler‐like reaction that
does not produce ROS (Allahverdiyeva et al., 2013), whereas
the heterodimer Flv2/4 is only present in β‐cyanobacteria and
was reported to be involved in photoprotection of both PSII
and PSI (Zhang et al., 2009, 2012; Bersanini et al.,
2014, 2017; Chukhutsina et al., 2015; Santana‐Sanchez et al.,
2019). In angiosperms, in which FDPs are absent, in-
troduction of two FDPs could therefore possibly replace
Table 1. Continued
Strategy and gene of interest
Targeted process
or protein complex Species
Phenotype
associated with
increased
biomass
accumulation
Phenotype
associated with
enhanced
abiotic stress
tolerance References
et al., 2020
Overexpression of
cyanobacterial flavodoxin
Electron transfer Tobacco — Iron deficiency,
oxidative stress
Tognetti et al.,
2006, 2007a, 2007b;
Zurbriggen et al., 2008;
Shvaleva et al.,
2009; Coba de la Peña
et al. 2010; Giró et al.,
2011; Ceccoli et al.,
2012; Lodeyro et al.,
2012; Gharechahi et al.,
2015; Mayta et al.,
2018; Gómez et al.,
2020; Niazian
et al., 2021
Overexpression of
cyanobacterial and
moss FDPs
AET Arabidopsis,
tobacco, barley, rice
Yes Drought/
fluctuating light
stress
Yamamoto et al.,
2016; Gómez et al.,
2018; Wada et al.,
2018; Tula et al., 2020;
Shahinnia et al., 2021;
Vicino et al., 2021
Abbreviations: AET, alternative electron transfer; ATP, adenosine triphosphate; LHC, light‐harvesting complex; NPQ, nonphotochemical quenching.
Optimizing light reactions to improve crop yield Journal of Integrative Plant Biology
580 February 2022 | Volume 64 | Issue 2 | 564–591 www.jipb.net
several ROS scavenging enzymes and reactions, thus
saving energy and nitrogen sources or adding extra pro-
tection. Indeed, transgenic lines of tobacco, Arabidopsis
and barley expressing cyanobacterial Flv1/3 or Flv2/4
proteins in chloroplasts showed that FDPs are able to act
as additional electron sinks in plants as well, particularly
under stress conditions, such as drought and fluctuating
light stress, thereby improving photosynthetic perform-
ance (Gómez et al., 2018; Tula et al., 2020; Shahinnia et al.,
2021; Vicino et al., 2021). In other instances, two FDPs
from the moss Physcomitrella patens were introduced into
Arabidopsis and rice, revealing similar effects of FDPs and
CET in maintaining the pmf under unfavorable growth
conditions (Yamamoto et al., 2016; Wada et al., 2018). In
summary, both sets of FDPs seem to fulfill similar roles
when expressed in angiosperms, even though they were
proposed to have different photoprotection targets (PSI vs.
PSII) in cyanobacteria. In one case, transgenic plants even
displayed improved biomass accumulation under non-
stress conditions (Tula et al., 2020), suggesting that FDPs
are promising tools for bioengineering of future crops
(Mullineaux, 2016).
Further cyanobacterial systems with potential for im-
proving photosynthetic efficiencies in plants are currently
under investigation and include the OCP and novel chlor-
ophylls with absorption wavelengths in the far‐red spectrum.
These red‐shifted chlorophylls d and f with absorption
maxima at 740 and 760 nm, respectively, have been dis-
covered in certain cyanobacterial species (Li and Chen,
2015). The novel chlorophyll d is predicted to be able to bind
to LHC proteins, expanding the range of light absorption of
chlorophyll a beyond 700 nm into the far‐red region, which is
often found in the lower canopy of plants and could therefore
enhance light harvesting and boost crop yields (Elias et al.,
2021). Cyanobacterial OCP, on the other hand, absorbs
wavelengths in the blue–green region and is attached to the
light‐harvesting antennae (phycobilisomes), where it is re-
sponsible for photoprotective dissipation of excess light en-
ergy as heat (NPQ) and scavenging of ROS (Muzzopappa and
Kirilovsky, 2020). This photoreceptor protein consists of an
effector N‐terminal domain (NTD), a sensor C‐terminal do-
main (CTD), and one ketocarotenoid (3‐hydroxyechinenone,
echinenone, canthaxanthin) or the xanthophyll zeaxanthin.
Upon absorption of strong blue–green light, the non-
covalently bound ketocarotenoid transfers from the CTD to
the NTD, thereby undergoing a conformational change and a
shift in color from orange to red, thus activating the
quenching state. OCP has not only been expressed in
ketocarotenoid‐producing microalgae for better solubilization
of the ketocarotenoids canthaxanthin and astaxanthin for
their use as nutraceuticals (Pivato et al., 2021), but is also
being exploited as a possible photoswitchable protein in
plants with implementations for plastid optogenetics, artificial
photosynthesis and synthetic biology due to its light‐
dependent conformational changes and uniqueness in cya-
nobacteria (Andreoni et al., 2017; Lechno‐Yossef et al.,
2017; Dominguez‐Martin and Kerfeld, 2019; Piccinini
et al., 2021).
CONCLUSIONS
“Photosynthesis: Ancient, essential, complex, diverse… and in
need of improvement in a changing world” (Niinemets et al.,
2016). This title of a conference summary article neatly de-
scribes the need for a shift in understanding photosynthesis
and its potential applications. Photosynthetic organisms have
optimized photosynthesis according to their needs, which in-
cludes survival and reproduction rather than enhanced yields.
Modern techniques of bioengineering, through synthetic biology
and gene editing, have provided useful means to targeting
specific features of metabolic pathways in a timelier manner
than conventional breeding does and are rapidly gaining trac-
tion in re‐engineering of photosynthesis (Zhu et al., 2020). Our
review has highlighted research demonstrating that it is pos-
sible to improve photosynthetic electron transport
(Figure 7; Table 1). Targeting the light‐harvesting antenna size is
a high potential approach in both plants as well as photo-
synthetic micro‐organisms, with a three‐fold increase in micro-
algae biomass (Negi et al., 2020) and 25%–40% more plant
biomass in transgenic tobacco andCamelina, respectively (Kirst
et al., 2017; Friedland et al., 2019). Furthermore, improving
photoprotective traits, such as NPQ relaxation and D1 photo-
protection, resulted in 15%–20% enhanced growth in field‐
grown plants (Kromdijk et al., 2016; Hubbart et al., 2018; Chen
et al., 2020). LET reactions are very well studied and gene ex-
pression manipulation of the Rieske‐FeS protein of the Cyt b6f
complex and the mobile electron carrier plastocyanin were re-
vealed as the most promising targets for boosting plant yields
(Simkin et al., 2017b; Pan et al., 2018). AET pathways in higher
plants, on the other hand, are less well understood, and ma-
nipulation of the putative components seems to affect stress
tolerance rather than enhance photosynthetic efficiency. In
contrast, introduction of alternative electron carriers and ac-
ceptors from lower plants, microalgae and cyanobacteria,
which often show higher efficiencies than their equivalents in
higher plants, could restore electron transfer pathways that
were lost in higher plants during the course of evolution and
improve plant growth (cyt c6: Chida et al., 2007; Yadav et al.,
2018; López‐Calcagno et al., 2020; FDPs: Tula et al., 2020). In
addition to the strategies discussed, combinations of different
approaches may be strongly synergistic and when translated
into staple crop species may allow an even greater boost in
photosynthetic efficiency and crop productivity.
ACKNOWLEDGEMENTS
This work was supported by the Realizing Increased Photo-
synthetic Efficiency (RIPE) project at the University of Illinois via
a subaward to Johannes Kromdijk. RIPE is possible through
support from the Bill & Melinda Gates Foundation, Foreign,
Optimizing light reactions to improve crop yieldJournal of Integrative Plant Biology
www.jipb.net February 2022 | Volume 64 | Issue 2 | 564–591 581
Commonwealth & Development Office, and the Foundation for
Food and Agriculture Research Grant No. OPP1172157.
CONFLICTS OF INTEREST
The authors declare there are no conflicts of interest.
Edited by: Zhizhong Gong, China Agricultural University, China
Received Oct. 29, 2021; Accepted Dec. 22, 2021; Published Dec.
28, 2021
OO: OnlineOpen
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